Description: Background: Molecular genetic testing is recommended for the diagnosis of many inherited diseases, and also informs prognosis and stratified therapy. However, access to diagnosis is often limited by cost and availability. Recently introduced high-throughput bench-top DNA sequencing platforms have the potential to overcome these limitations. Methodology/principal findings: Here we evaluated two next-generation sequencing platforms for molecular diagnostics; MiSeq (Illumina) and Ion Torrent PGM (Life Technologies), using upstream microfluidic multiplex PCR target-enrichment. We sequenced six genes (KCNE1, KCNE2, KCNH2, KCNQ1, RYR2 and SCN5A) associated with important inherited cardiac conditions in a panel of well-characterised human DNA samples that contained 1256 previously reported disease-associated variants on both platforms. The MiSeq produced a higher proportion of high-quality, on-target reads, but coverage was less even. Overall, 97.9% of the target was sequenced adequately for variant calling on the MiSeq, and 96.8% on the PGM. Regions missed tended to be of high GC-content (>80%), and most were problematic for both platforms, though some GC-independent drop-out was also seen on the MiSeq. For captured regions (96.9%, MiSeq; 95.5% PGM), sensitivity (100%, MiSeq; 99.1%, PGM) and positive predictive value (PPV) (95.9%, MiSeq; 95.5%, PGM) were excellent when compared with Sanger sequencing. As compared to one MiSeq run (1.2Gbases of sequence), three PGM runs of greater technical difficulty and of higher overall expense were required. Conclusions/significance: MiSeq and Ion Torrent PGM have similar high sensitivities and PPVs for variant detection, and coupled with target enrichment solutions provide a mature platform for the molecular diagnosis of inherited diseases. In the short-term, conventional Sanger sequencing may be used as an adjunct to interrogate 2-3% of the protein-coding bases missed by these methods. Though there are minor performance differences at this throughput, platforms differ primarily in terms of cost, scalability and ease of use.

Data type: Other

Sample scope: Monoisolate

Release date: 2013-04-12

Last updated: 2013-04-13